Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. In addition to copying the DNA, all of the epigenetic information encoded in DNA methylation and histone modifications must also be copied. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Cells employ multiple repair and signaling responses to replication stress depending on the type, persistence, and location of the problem. We have employed a new method that my lab developed called iPOND (isolation of proteins on nascent DNA) to study DNA replication, chromatin deposition and maturation, and the replication stress response. When combined with quantitative mass spectrometry, this method is particularly powerful at characterizing changes in replisome composition in response to perturbations. As an unbiased approach, it is also a discovery tool. In preliminary studies we identified several new proteins that function at active and damaged replication forks. This application focuses on two of these proteins, which we found to be involved in the replication stress response and in re- establishment of epigenetic information during the cell division cycle. Aim 1 uses genetic and biochemical methods to test specific hypotheses and models about how these proteins maintain genome and epigenome stability. Aim 2 examines how replication forks deal with DNA-protein crosslinks including those caused by common environmental toxins and cancer therapies. How a cell deals with these types of fork-stalling events is poorly characterized, and our quantitative iPOND-mass spectrometry approach provides a unique discovery opportunity to understand this significant threat to genome integrity. Since cancer cells have elevated levels of replication stress and many cancer therapeutics work by interfering with DNA replication, these studies will also generate discoveries that may be translated into the cancer clinic.